School of Medicine, Shandong University, Jinan, 250100, China.
2 Department of Hematology, The First Affiliated Hospital of University of Science and Technology of China, Hefei, 230001, China.
3 The First Hospital Affiliated to Anhui University of Traditional Chinese Medicine, Hefei, 230001, China.
4 Department of Laboratory Medicine, The First Affiliated Hospital of University of Science and Technology of China, Hefei, 230001, China.
5 University of Sassari, Sassari, Italy.
6 School of Computer Science, University of Science and Technology of China, Hefei, 230001, China.
7 School of clinical medical, University of medical of Anhui, Hefei, 230001, China.
Received: July 12, 2020
Accepted: February 3, 2020
Mediterr J Hematol Infect Dis 2020, 12(1): e2020012 DOI 10.4084/MJHID.2020.012
| This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Micro (mi) RNAs play an important role in the pathogenesis and
development of acute myeloid leukemia (AML), and their abnormal
expression may be sufficient to predict the prognosis and outcomes in
AML patients. We evaluated the clinical diagnostic value of
miRNA-181a-3p in predicting prognosis and outcomes in patients with AML.
Micro (mi)RNAs are small, noncoding RNAs that bind their target mRNAs and inhibit the expression of encoded proteins. MiRNAs have critical biological functions, including in hematopoietic cell proliferation, differentiation, and apoptosis, and may also play an essential role in the pathogenesis and development of AML. Several studies have identified that distinctive miRNA profiles are associated with cytogenetic subtypes, mutations, and clinical outcomes of AML.[3-5] For example, decreased miR-196b expression is associated with the absence of FLT3-ITD and NPM1 mutations, and high miR-196b expression acts as a predictive factor of poor prognosis.[4-5] Therefore, miRNA expression levels may be suitable to predict prognosis and outcomes in AML patients.
The miR-181 family is thought to be involved in a number of biological processes, including transcription, translation, and signaling transduction. In humans, the miR-181 family has four mature homologs (hsa-miR-181a, hsa-miR-181b, hsa-miR-181c and hsa-miR-181d). MiR-181a-3p belongs to miR-181a mature homologs, acts as a negative post-transcriptional regulator of Nuclear Factor kappa-B (NF-κB) signaling pathway by directly targeting NF-κB essential modulator (NEMO/IKBKG) in Human Umbilical Vein Endothelial Cells (HUVECs). Our previous study showed that abnormal expression of miR-181a-3p was associated with human monocytic leukaemia cell line THP-1 cell. However, few studies have focused on the clinical role of miR-181a-3p in AML patients. Therefore, the present study examined the expression of miR-181a-3p in AML patients prior to treatment to evaluate its clinical diagnostic value and predictive role in the prognosis and outcomes of AML patients.
Materials and Methods
TCGA data set. Publicly available clinical, gene expression data for 188 patients with AML were downloaded from TCGA data portal.
Data analysis. We used SPSS (version 24), GraphPad Prism (version 7) software and R language to analyze the data. The unpaired Student’s t test (two-tailed) was performed to compare differences in miRNA expression between different groups. Chi-square tests were used to test the association between the expression of miRNAs and clinicopathological characteristics. Cox proportional hazards models were used to analyze the prognostic utility of miRNA expression for disease-free survival (DFS) and overall survival (OS) in AML patients.
|Table 1. Clinical characteristics of AML patients at diagnosis.|
The clinical value of miRNA-181a-3p in the diagnosis of AML. To examine whether miRNA-181a-3p was abnormally expressed in patients with AML, we detected miRNA expression in 60 healthy controls and 119 adult patients with newly diagnosed AML. Compared with healthy controls, the expression of miRNA-181a-3p (P<0.001, Figure 1 A) was significantly increased in AML patients, and in the samples of M1, M2, M3, and M4 subtypes (Figure 1 B). Furthermore, we compared the expression of microRNAs in 4 subtypes base on molecular genetic abnormalities. Compared with healthy controls, the expression of miRNA-181a-3p was significantly increased in all four subtypes (Figure 1 C).
To assess the clinical diagnostic value of miR-181a-3p in discriminating AML patients from healthy controls, we performed receiver operating characteristic (ROC) curve analyses. The Area Under Curve (AUC) of miR-181a-3p was (0.654, 95% CI, 0.575 to 0.732, P<0.001, Figure 1D). Compared with health controls, miR-181a-3p showed significant difference in the samples of FAB M1, M2, M3 subtypes (Figure 1E) and PML/RARa, AML1/ETO subtypes (Figure 1F) compared with controls, suggesting that it had value in discriminating patients from controls. The AUC of miR-181a-3p in the samples of different subtypes was showed in Table 2.
|Table 2. The AUC of miR-181a-3p in the samples of different subtypes. P-values calculated by Unpaired student's t test. AUC = The Area Under Curve. 95% CI = 95% confidence interval.|
The expression of miRNA-181a-3p was decreased when patients achieved CR after induction chemotherapy. To examine whether miRNA-181a-3p was decreased in patients who achieved CR after induction chemotherapy, we detected its expression from blood specimens in 10 patients. On day 28 after induction chemotherapy, miRNA-181a-3p expression was significantly decreased in 80% of patients (P<0.001, Figure 2). Moreover, on day 28 after induction chemotherapy, miR-181a-3p was expressed at higher levels compared with healthy controls (P<0.05, Figure 2A).
Association of miR181a-3p expression with AML patient outcome. A total of 119 adult patients with newly diagnosed AML were recruited, 8 patients died within 30 days after chemotherapy, finally 111 patients were included for statistical analysis. A total of 81 patients achieved complete remission (CR), whereas 30 patients failed to. To graphically display the association of miR181a-3p expression with CR achievement, we compared expression levels in patients who achieved CR (n=81) with those who failed to achieve CR (n=30) (Fig 3A). At the time of diagnosis, miRNA-181a-3p expression level was correlated with the response to induction chemotherapy (P<0.05).
According to miR-181a-3p expression, patients with AML were dichotomized into high (above median expression levels) and low (below or at median expression levels) groups. Kaplan–Meier survival curves showed that patients with higher miR-181a-3p expression levels at diagnosis presented with a better OS (P=0.014, Fig 3D) , but not a better DFS (P=0.062, Fig 3C), than those with lower expression levels. In the TCGA analysis, AML patients with higher miR-181a-3p also presented with a better OS (P=0.008, Fig 3E).
The Mantel–Cox test and Gehan–Breslow–Wilcoxon test were performed to determine the relationship between miR-181a-3p expression and OS or DFS (Fig 3B). The Gehan–Breslow–Wilcoxon test highlighted the prognostic value of increased miR-181a-3p expression at diagnosis both for disease relapse (HR: 1.597; 95% CI: 0.9804-2.559; P=0.03) and death (HR: 2.062; 95% CI: 1.160-3.456; P=0.02). Thus, a higher miR-181a-3p expression at diagnosis was significantly associated with patient outcome.
Finally, multivariate Cox analysis was performed to determine the relationship between independent prognostic value and OS or DFS. The multivariate Cox analysis was adjusted for patients’ age, gender, WBC count and disease risk stratification. Multivariate analysis (Table 3) highlighted the of miR-181a-3p levels on AML diagnosis not for disease relapse (P>0.05) but for death (HR: 1.923; 95% CI: 1.035-3.574; P<0.05).
|Table 3. Multivariate Cox regression analysis of independent risk factors influencing OS and DFS. P-values calculated by Unpaired student's t test. WBC = white blood cell. 95% CI = 95% confidence interval.|
Assessment of the relationship between miR-181a-3p ectopic expression and IKBKG and NF-κB family. Since miR-181a-3p blocks the NF-κB signaling pathway by targeting NEMO/IKBKG in Human Umbilical Vein Endothelial Cells (HUVECs), whether IKBKG and NF-κB expressions are affected by miR-181a-3p in AML cells needed to be determined. NF-κB family contains NF-κB1, NF-κB2, RelA, RelB and Rel. Firstly, we investigated the relation of miR-181a-3p ectopic expression with NEMO/IKBKG and NF-κB family in a set of primary TCGA AML patients. NEMO/IKBKG expression was positively correlated with expression of NF-κB family (NF-κB1, NF-κB2, RelA, RelB) (Person correlation= 0.377, P<0.01; Person correlation= 0.598, P<0.01; Person correlation= 0.557, P<0.01; Person correlation= 0.524, P<0.01). MiR-181a-3p expression was negatively correlated with expression of NF-κB family (NF-κB1, NF-κB2, RelA, RelB) (Person correlation= -0.209, P<0.05; Person correlation= -0.555, P<0.01, Person correlation= -0.19, P<0.05, Person correlation= -0.309, P<0.01) and NEMO/IKBKG (Person correlation= -0.313, P<0.01). Then we investigated the relation of miR-181a-3p expression with NEMO/IKBKG and NF-κB in 59 AML patients, and found that miR-181a-3p expression was negatively correlated with the expression of NF-κB (Person correlation= -0.2795, P=0.0321) and NEMO/IKBKG (Person correlation= -0.2613, P=0.0456). The linear correlation analysis in AML samples were showed in Table 4..
|Table 4. The linear correlation analysis in AML samples. P-values calculated by Pearson's Correlation. 95% CI = 95% confidence interval.|
In recent years, novel high-throughput sequencing techniques have significantly advanced our understanding of the molecular pathogenesis of AML.[10-11] Several recurrent mutations in genes encoding epigenetic modifiers have been identified that affect not only disease phenotype but also a response to therapy.[12-13] MiRNAs play an important role in the pathogenesis and development of AML, and their abnormal expression is associated with specific cytogenetic subsets or mutations of AML, suggesting that they could be used as independent biomarkers for determining the outcomes of AML patients.[4,14-15]
MiR-181a belongs to the miR-181 family, its role in tumors is still controversial, and it may function as a tumor promoter or suppressor depending on tumor type. In hematologic malignancies, miR-181a functions as a tumor suppressor in cellular division and differentiation. AML patients with higher miR-181 expression at diagnosis have a better prognosis than those with lower miR-181 expression, miR-181 may be a diagnostic biomarker and predictor of prognosis in AML patients.[17-19]
Precursor miR-181a can be processed into two mature strands: miR-181a-3p and miR-181a-5p. MiR-181a-3p is highly expressed in RPMI8226 cell-derived extracellular vesicles (R-EVs) and regulates cell proliferation. Mir-181a-3p blocks the NF-κB signaling pathway by targeting NEMO/IKBKG in Human Umbilical Vein Endothelial Cells (HUVECs), and miR-181a-3p mimics treatment prevents myeloid cell recruitment and decreased the expression of TNF-α in apoE −/− mice.8 NF-κB is an important transcription factor, which plays a crucial cancer-promoting role in Acute myeloid leukemia (AML).[21-22] It has been known that chromosomal translocations or gene mutations leading to the increase in NF-κB activity. NEMO/IKBKG acts as a crucial antiapoptotic transcription factor, which is crucial for the activation of NF-κB. NF-κB family contains RelA, RelB, NF-κB1, NF-κB2 and Rel. We found that miR-181a-3p expression was negatively correlated with the expression of NF-κB family (NF-κB1, NF-κB2, RelA, RelB) and NEMO/IKBKG in TCGA samples. In our study, miR-181a-3p expression was negatively correlated with the expression of NF-κB and NEMO/IKBKG in 59 AML patients. Maybe mir-181a-3p affects AML cell proliferation and apoptosis by targeting NEMO/IKBKG. We should make more efforts to test this hypothesis.
In summary, we reported a clinical role for miR-181a-3p in AML patients for the first time. MiRNA-181a-3p expression was shown to have value in discriminating AML patients from healthy controls, and to correlate with the response to induction chemotherapy. Patients with higher miR-181a-3p expression levels at diagnosis demonstrated an improved OS. Therefore, the miR-181a-3p expression may be an independent prognostic biomarker for AML patient outcomes.
- Coombs CC, Tallman MS, Levine RL: Molecular therapy
for acute myeloid leukaemia. Nature Reviews Clinical Oncology 2016,
13(5):305-318. https://doi.org/10.1038/nrclinonc.2015.210 PMid:26620272 PMCid:PMC5525060
G, Mrozek K, Radmacher MD, Garzon R, Bloomfield CD: The prognostic and
functional role of microRNAs in acute myeloid leukemia. Blood 2011,
117(4):1121-1129. doi:10.1182/blood-2010-09-191312. https://doi.org/10.1182/blood-2010-09-191312 PMid:21045193 PMCid:PMC3056468
JA, O'Connell RM: MicroRNAs and acute myeloid leukemia: therapeutic
implications and emerging concepts. Blood 2017, 130(11):1290-1301. https://doi.org/10.1182/blood-2016-10-697698 PMid:28751524 PMCid:PMC5600138
M, Brunet S, Nomdedeu J, Tejero R, Diaz T, Pratcorona M, Tormo M,
Ribera JM, Escoda L, Duarte R et al: MicroRNA expression at diagnosis
adds relevant prognostic information to molecular categorization in
patients with intermediate-risk cytogenetic acute myeloid leukemia.
Leukemia 2014, 28(4):804-812. https://doi.org/10.1038/leu.2013.281 PMid:24072101
MK, Chiu YC, Chou WC, Hou HA, Chuang EY, Tien HF: A 3-microRNA scoring
system for prognostication in de novo acute myeloid leukemia patients.
Leukemia 2015, 29(5):1051-1059. https://doi.org/10.1038/leu.2014.333 PMid:25428263
Z, Wan X, Gu Z, Zhang H, Yang X, He L, Miao R, Zhong Y, Zhao H:
Evolution of the mir-181 microRNA family. Computers in Biology and
Medicine 2014, 52:82-87. https://doi.org/10.1016/j.compbiomed.2014.06.004 PMid:25016292
R, Lin HS, Zhang XH, Yin XL, Ning HM, Liu B, Zhai PF, Gong JN, Shen C,
Song L et al: MiR-181 family: regulators of myeloid differentiation and
acute myeloid leukemia as well as potential therapeutic targets.
Oncogene 2015, 34(25):3226-3239. https://doi.org/10.1038/onc.2014.274 PMid:25174404
Y, Yuan J, Zhang F, Lei Q, Zhang T, Li K, Guo J, Hong Y, Bu G, Lv X et
al: MicroRNA-181a-5p and microRNA-181a-3p cooperatively restrict
vascular inflammation and atherosclerosis. Cell Death & Disease
2019, 10(5):365. https://doi.org/10.1038/s41419-019-1599-9 PMid:31064980 PMCid:PMC6504957
XX, Zhang SS, Dai CY, Peng J, Pan Q, Xu LF, Ma XL: LukS-PV-Regulated
MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and
Apoptosis by Down-Regulating NF1 and Bcl-2. Cellular physiology and
biochemistry. International Journal of Experimental Cellular
Physiology, Biochemistry, and Pharmacology 2017, 44(3):1093-1105. https://doi.org/10.1159/000485415 PMid:29179212
L, Dohner K, Dohner H: Genomics of Acute Myeloid Leukemia Diagnosis and
Pathways. Journal of clinical oncology : official journal of the
American Society of Clinical Oncology 2017, 35(9):934-946. https://doi.org/10.1200/JCO.2016.71.2208 PMid:28297624
- Ferrando AA, Lopez-Otin C: Clonal evolution in leukemia. Nature Medicine 2017, 23(10):1135-1145. https://doi.org/10.1038/nm.4410 PMid:28985206
- Short NJ, Rytting ME, Cortes JE: Acute myeloid leukaemia. Lancet (London, England) 2018, 392(10147):593-606. https://doi.org/10.1016/S0140-6736(18)31041-9
- Medinger M, Passweg JR: Acute myeloid leukaemia genomics. British Journal of Haematology 2017, 179(4):530-542. https://doi.org/10.1111/bjh.14823 PMid:28653397
Leeuw DC, Verhagen HJ, Denkers F, Kavelaars FG, Valk PJ, Schuurhuis GJ,
Ossenkoppele GJ, Smit L: MicroRNA-551b is highly expressed in
hematopoietic stem cells and a biomarker for relapse and poor prognosis
in acute myeloid leukemia. Leukemia 2016, 30(3):742-746. https://doi.org/10.1038/leu.2015.160 PMid:26108690
G, Radmacher MD, Mrozek K, Bloomfield CD: MicroRNA expression in acute
myeloid leukemia. Current Hematologic Malignancy Reports 2009,
4(2):83-88. https://doi.org/10.1007/s11899-009-0012-7 PMid:20425419
- Roth E, Cao J: MiR-181 suppresses metastasis via MMP-14. Aging 2015, 7(10):740-741. https://doi.org/10.18632/aging.100824 PMid:26527609 PMCid:PMC4637197
S, Maharry K, Radmacher MD, Mrozek K, Holland KB, Margeson D, Whitman
SP, Hickey C, Becker H, Metzeler KH et al: Prognostic significance of
expression of a single microRNA, miR-181a, in cytogenetically normal
acute myeloid leukemia: a Cancer and Leukemia Group B study. Journal of
clinical oncology. Official Journal of the American Society of Clinical
Oncology 2010, 28(36):5257-5264. https://doi.org/10.1200/JCO.2010.29.2953 PMid:21079133 PMCid:PMC3018359
A, Rybka J, Baczynska D, Poreba R, Mazur G, Kuliczkowski K: Expression
of microRNA-181 determines response to treatment with azacitidine and
predicts survival in elderly patients with acute myeloid leukaemia.
Oncology Letters 2016, 12(4):2296-2300. https://doi.org/10.3892/ol.2016.4970 PMid:27698792 PMCid:PMC5038519
H, Lal K, Yang FF, Chen J: The pathological role and prognostic impact
of miR-181 in acute myeloid leukemia. Cancer Genetics 2015,
208(5):225-229. https://doi.org/10.1016/j.cancergen.2014.12.006 PMid:25686674 PMCid:PMC4466067
L, Lei Q, Wang H, Xu C, Liu T, Kong F, Yang C, Yan G, Sun L, Zhao A et
al: Tumor-derived extracellular vesicles inhibit osteogenesis and
exacerbate myeloma bone disease. Theranostics 2019, 9(1):196-209. https://doi.org/10.7150/thno.27550 PMid:30662562 PMCid:PMC6332790
Y, Yoshimi A, Kataoka K, Nakagawa M, Kumano K, Arai S, Kobayashi H,
Saito T, Iwakura Y, Kurokawa M: Positive feedback between NF-kappaB and
TNF-alpha promotes leukemia-initiating cell capacity. The Journal of
Clinical Investigation 2014, 124(2):528-542. https://doi.org/10.1172/JCI68101 PMid:24382349 PMCid:PMC3904603
MC, Schuringa JJ, Vellenga E: Constitutive NF-kappaB activation in AML:
Causes and treatment strategies. Critical reviews in
oncology/hematology 2016, 98:35-44. https://doi.org/10.1016/j.critrevonc.2015.10.001 PMid:26490297
S, Ising C, Barrera Aranda B, Hohne M, Schermer B, Benzing T,
Brinkkoetter PT: The NF-kappaB essential modulator (NEMO) controls
podocyte cytoskeletal dynamics independently of NF-kappaB. American
Journal of Physiology Renal Physiology 2015, 309(7):F617-626. https://doi.org/10.1152/ajprenal.00059.2015 PMid:26268269